Download Dr Joanne Chory of The Salk Institute, Howard Hughes Medical

How do light signals control nuclear
genes for leaf & plastid development?
Can divide into 3 basic steps (or parts):
1. Receiving the signal (photoreceptors)
2. Transmitting (and amplifying?) the signal
to the nucleus
3. Activating (de-repressing?) or repressing
transcription of genes associated with
“greening” or “de-etiolation”
Transcriptional control of the pea rbcS3 gene by
light:: The molecular approach
• Comparative analysis of 5’-upstream sequences of the rbcS gene
family (pea) identified light-responsive elements (LRE)
Enhancer
______________________________________
III* II* I II III
-330
VI
-50
V
+1
N-H. Chua
• Several putative trans-acting factors for this promoter were identified based
on their in vitro ability to bind to specific elements
- GT1, AF2 & AF3 binds to, or near, boxes II and/or III (and II* and/or III*)
- AF1 binds box VI
• Present in both light and dark, however.
• Some maybe regulated by phosphorylation-dephosphorylation
- Binding of AF3 to DNA is promoted by phosphorylation
- Kinase may be a casein kinase 2 (CK2)
Postive and negative factors from a genetic
approach in Arabidopsis
Another long hypocotyl mutant,
Hy5, lacks a bZIP factor that
promotes transcription from a
number of genes with LREs
- Hy5 also responds to the
blue (Cry) and red light (Phy)
photoreceptors
bZIP proteins have basic (+) DNAbinding domain and a leucine
dimerization zipper
COP/DET genes: Pleiotropic repressors of leaf and
plastid development
• Pleiotropy- one gene affects many processes
• J. Chory identified a mutant DET1 that de-etiolates
even in darkness
– cotyledons are not green, but they do expand
– plastids develop into chloroplast-like organelles
– Many greening genes (rbcS and cab) are on
Det1 light
Det1 dark
WT dark
COP/DET/FUS genes: Pleiotropic repressors of leaf
and plastid development
COP (constitutive
photomorphogenesis) and FUS
mutants similar to DET1
10 COP genes:
- COP1, key repressor of photomorph.
- 8 are subunits of a large complex,
COP9 signalosome,
- COP1 and COP9 complex also
found in animal cells
COP1
- COP1 is a ubiquitin ligase, which
triggers degradation of transcription
factors (HY5) by the proteasome, also
interacts with COP9 and DET1
Yi and Deng, 2005, TCB 15:618
PhyA, PhyB, Cry1, Cry2 inhibit COP1-mediated
degradation of transcription factors that activate
photomorph. genes
Cry proteins are
activated by BL,
phosphorylated,
and then bind to
COP1
COP1 also
moves to
cytoplasm in
light.
How do PhyA and PhyB inhibit COP1??
Phytochrome Interacting
Factors (PIFs)
Another Model for Phytochrome Action.
Enamul Huq, UT
Peter Quail, USDA
PhyB sees Red and PhyA, Far-red
D
Rc
Arabidopsis
thaliana
mutants
FRc
WT
phyA phyB
Tepperman et al., 2004
Two-Hybrid Screening Strategy to get Phy
Interacting Factors (PIFs)
phy PIFs AD
BD
GAL1 UAS
HIS3/lacZ
PIF3 (bHLH protein): Required for Full PhyBMediated Greening Response
Pho4 (bHLH) bound to DNA
Monte et al (2004) PNAS
CCA1 & LHY have G-box
Martinez-Garcia et al (2000) Science 288: 859
At least some PhyA and PhyB Translocate into
Nucleus in Light
phyB
phyA
Nagy and Schaefer (2000) EMBO J. 19: 157-163.
GFP-tagged Phy proteins
Postulated Direct Targeting of Light Signal
to Promoter-Bound PIF 3
PrB
R
PIF3 PIF3
G-box
TA TA
FR
PfrB
PfrB
FR
PrB
NUCLEUS
PfrB
PIF3 PIF3
G-box
CYTOPLASM
PIC
TA TA
Does PIF3 heterodimerize?
A 2-Hybrid Screen with PIF3 as Bait
PIF3
BD
Y AD
HIS3/lacZ
GAL1 UAS
** PIF1 and PIF4 are new bHLH proteins
What is/are the function of the heterodimer(s)?
SUMMARY
1. Interaction of DNA-bound PIF3 with Pfr form of
PhyB may provide for direct regulation of gene
expression in response to light.
2. PIF3 helps controls greening process, and interacts
with other PIFs.
3. Phy signaling involves direct interaction with
transcription factors (PIFs).